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Abstract:

The present invention relates to a compound comprising the third
Immunoglobulin (Ig3) module, and/or the fourth Immunoglobulin (Ig4)
module, and/or the fifth immunoglobulin (Ig5) module, and/or the first
Fibronectin III (Fn3,1) module, and/or the second Fibronectin III (Fn3,2)
module of neural cell adhesion molecule (NCAM), or a fragment, or a
variant thereof, capable of interacting with an Fibroblast Growth Factor
(FGF) receptor and/or Adenosine-Tri-Phosphate (ATP) and/or L1, and
thereby the compounds are capable of inducing differentiation, modulating
proliferation, stimulate regeneration, neuronal plasticity and/or
survival of cells. Further, the present invention relates to a
pharmaceutical composition comprising said compound, a process of
producing a pharmaceutical composition and the use of said compound.

Claims:

1. A compound which is(I) a peptide consisting of 5 to about 100 amino
acid residues, said peptide comprising(i) the second Fibronectin III
(Fn3,2) module of the neural cell adhesion molecule (NCAM) Swiss Prot
P13591, or a fragment at least five amino acids long thereof, or a
substitution variant of said module or fragment wherein said variant is
at least 75% identical to said Fn3,2 module or said fragment, and/or(ii)
the first Fibronectin III (Fn3,1) module of NCAM, or a fragment at least
five amino acids long thereof, or a substitution variant of said module
or said fragment, wherein said variant is at least 75% identical to said
fragment,(II) an oligomeric peptide consisting essentially of two or more
monomers, which may be the same or different, and which independently
consist of an amino acid sequence satisfying that of a peptide according
to (I), or(III) a salt, ester or amide of the peptide of (I) or the
oligomeric peptide of (II),said compound being in at least partially
purified form.

2. The compound of claim 1 which is capable of interacting with a
fibroblast growth factor (FGF) receptor.

3. The compound according to claim 1, comprising the second Fn 3,2 module
of NCAM, or a fragment, or a variant thereof.

4. The compound according to claim 1, wherein (ii) comprises an amino acid
sequence of the formulaL1-A-L2-B-L3-C-L4, whereinone of A, B, C is
selected from a basic amino acid,one of A, B, C is selected from a
hydrophobic amino acid,one of A, B, C is glycine, andL1, L2, L3, and L4
are each independently an amino acid sequence having n amino acid
residues, wherein n is independently an integer of from 0 to 5.

5. The compound according to claim 4, wherein B is glycine, A is a basic
amino acid residue, and C is a hydrophobic amino acid residue.

6. The compound according to claim 5, wherein A is lysine (K), and C is
leucine (L) or alanine (A).

8. The compound according to claim 1, wherein (i) comprising an amino acid
sequence of the formulaL1-A-L2-B-L3-C-L4-D-L5, whereinone of A, B, C, D
is selected from a basic amino acid residue,one of A, B, C, D is selected
from a hydrophobic amino acid residue,one of A, B, C, D is selected from
an acidic amino acid residue,one of A, B, C, D is glycine, andL1, L2, L3,
L4 and L5 are each independently an amino acid sequence having n amino
acid residues, wherein n is independently an integer of from 0 to 5.

9. The compound according to claim 1, wherein said compound comprises a
peptide homologous to the FG loop of the Fn3,2 module of the NCAM
molecule, or a fragment, or a variant thereof.

10. The compound according to claim 9, wherein said compound comprises a
peptide sequence having the motif A-E-N-Q-X-X-K, wherein X may be any
amino acid residue.

13. The compound according to claim 8, wherein the amino acid sequence has
the formulaAENQ-L4-G (SEQ ID NO:205).

14. The compound according to claim 1, wherein (i) comprising an amino
acid sequence of the formulaA-B-L3-L4-C-L4, whereinA is a hydrophobic
amino acid residue,B is an acidic amino acid residue,L3 is one or more
hydrophilic amino acid residue(s),L4 is an amino acid sequence of 1 to 5
amino acid residues, andC is glycine.

15. The compound according to claim 1 comprising a sequence of the formula
L1-A-L2-B-L3-C-L4-D-L5-E-L6, wherein L1, L2, L3, L4, L5 and L6 are each
independently an amino acid sequence having n residues, where n is
independently an integer of from 0 to 5,at least one of L1, L2, L3, or L4
comprises the amino acid residue Y and one of the other comprises the
amino acid residue K,L5 and/or L6 individually is K, and A, B, C, D, and
E are any amino acid, with the proviso, that the smallest distance
between a Y and a K is at least 5 amino acids.

16. The compound according to claim 1, wherein the compound is a dimer or
a multimer, and thereby comprises a plurality of monomers.

17. The compound according to claim 16, wherein the monomers are identical
to each other.

18. The compound according to claim 16, wherein the monomers are not
identical to each other.

19. The compound according to claim 1, consisting of 10-90 amino acid
residues.

20. The compound according to claim 1, consisting of 5-20 amino acid
residues.

21. A pharmaceutical composition comprising at least one compound as
defined in claim 2.

22. The pharmaceutical composition according to claim 21, wherein the
compound is the NCAM Fn3,2 module, or a fragment thereof, or a variant
thereof.

23. The pharmaceutical composition according to claim 21, wherein the
compounds are formulated as dimers.

24. The pharmaceutical composition according to claim 21, wherein the
compounds are formulated as multimers.

26. The compound of claim 1 wherein the peptide of (I) or all of the
corresponding monomeric peptide units of (II), is or are said module or
said fragment of said module.

27. The compound of claim 1, wherein said sequence identity of at least
75% is at least 80%.

28. The compound of claim 1, wherein said sequence identity of at least
75% is at least 85%.

29. The compound of claim 1, wherein said variant differs from said module
or from said fragment, if at all, solely by one or more conservative
substitutions.

30. The compound of claim 27, wherein said variant differs from said
module or from said fragment, if at all, solely by one or more fragment
of (i) or from said conservative substitutions.

31. The compound of claim 28, wherein said variant differs from said
module or from said fragment, if at all, solely by one or more
conservative substitutions.

32. The compound of claim 1, wherein said variant differs from said module
or from said fragment, if at all, solely by one or more conservative
substitutions, and/or by replacement of an L-amino acid with the
corresponding D-amino acid.

33. The compound of claim 1 wherein the peptide of (I), and the
corresponding monomer of the oligomeric peptide of (II), are at least
seven amino acid residues.

34. The compound of claim 1 wherein the peptide of (I), and the
corresponding monomer of the oligomeric peptide of (II), are at least ten
amino acid residues.

35. The compound of claim 1 wherein the peptide of (I) is either a
fragment, consisting of at least five consecutive amino acids of the FG
loop of the module of (i) or (ii), or a variant of such a fragment, and
the monomers of the oligomeric peptide of (II) each consist independently
of amino acid sequences of such fragments and variants.

36. The compound of claim 1 wherein the peptide of (I) is either a
fragment, consisting of at least seven consecutive amino acids of the FG
loop of the module of (i) or (ii), or a variant of such a fragment, and
the monomers of the oligomeric peptide of (II) each consist independently
of amino acid sequences of such fragments and variants.

37. The compound of claim 31 wherein the peptide of (I) is either a
fragment, consisting of at least seven consecutive amino acids of the FG
loop of the module of (i) or (ii), or a variant of such a fragment, and
the monomers of the oligomeric peptide of (II) each consist independently
of amino acid sequences of such fragments and variants.

38. The compound of claim 1, which is in pharmaceutically acceptable form.

39. The compound of claim 1, which is in soluble form.

40. The compound of claim 1, wherein the compound is a compound according
to (I), or a ester, salt of amide of a compound according to (I).

41. The compound of claim 1, wherein the multimeric peptide of (II) is a
dendrimer.

42. The compound of claim 1, wherein the peptide of (I) consists of 5-20
residues.

43. The compound according to claim 2, wherein the FGF receptor is
selected from the group consisting of FGF receptor I, FGF receptor II,
FGF receptor III, and FGF receptor IV.

44. The compound of claim 43 in which the receptor is a human FGF
receptor.

45. The compound of claim 43 in which the receptor is a human FGF
receptor.

46. The compound according to claim 43, wherein (ii) comprises an amino
acid sequence of the formulaL1-A-L2-B-L3-C-L4, whereinone of A, B, C is
selected from a basic amino acid,one of A, B, C is selected from a
hydrophobic amino acid,one of A, B, C is glycine, andL1, L2, L3, and L4
are each independently an amino acid sequence having n amino acid
residues, wherein n is independently an integer of from 0 to 5.

47. The compound according to claim 43, wherein (i) comprising an amino
acid sequence of the formulaL1-A-L2-B-L3-C-L4-D-L5, whereinone of A, B,
C, D is selected from a basic amino acid residue,one of A, B, C, D is
selected from a hydrophobic amino acid residue,one of A, B, C, D is
selected from an acidic amino acid residue,one of A, B, C, D is glycine,
andL1, L2, L3, L4 and L5 are each independently an amino acid sequence
having n amino acid residues, wherein n is independently an integer of
from 0 to 5.

48. The compound according to claim 43, wherein said compound comprises a
peptide homologous to the FG loop of the Fn3,2 module of the NCAM
molecule, or a fragment, or a variant thereof.

49. The compound according to claim 43, wherein said compound comprises a
peptide sequence having the motif A-E-N-Q-X-X-K, wherein X may be any
amino acid residue.

50. The compound of claim 43, wherein said sequence identity of at least
75% is at least 80%.

51. The compound of claim 43, wherein said sequence identity of at least
75% is at least 85%.

52. The compound of claim 50, wherein said variant differs from said
module or from said fragment, if at all, solely by one or more fragment
of (i) or from said conservative substitutions.

53. The compound of claim 51, wherein said variant differs from said
module or from said fragment, if at all, solely by one or more
conservative substitutions.

54. The compound of claim 43 wherein the peptide of (I) is either a
fragment, consisting of at least five consecutive amino acids of the FG
loop of the module of (i) or (ii), or a variant of such a fragment, and
the monomers of the oligomeric peptide of (II) each consist independently
of amino acid sequences of such fragments and variants.

55. The compound of claim 43 wherein the peptide of (I) is either a
fragment, consisting of at least seven consecutive amino acids of the FG
loop of the module of (i) or (ii), or a variant of such a fragment, and
the monomers of the oligomeric peptide of (II) each consist independently
of amino acid sequences of such fragments and variants.

56. The compound according to claim 55, wherein the amino acid sequence
has the formulaAENQ-L4-G (SEQ ID NO:205)wherein L4 is null or an amino
acid sequence consisting of 1-5 amino acid residues.

57. The compound according to claim 43, wherein (i) comprising an amino
acid sequence of the formulaA-B-L3-L4-C-L4, whereinA is a hydrophobic
amino acid residue,B is an acidic amino acid residue,L3 is one or more
hydrophilic amino acid residue(s),L4 is an amino acid sequence of 1 to 5
amino acid residues, andC is glycine.

58. The compound according to claim 43 comprising a sequence of the
formula L1-A-L2-B-L3-C-L4-D-L5-E-L6, wherein L1, L2, L3, L4, L5 and L6
are each independently an amino acid sequence having n residues, where n
is independently an integer of from 0 to 5,at least one of L1, L2, L3, or
L4 comprises the amino acid residue Y and one of the other comprises the
amino acid residue K,L5 and/or L6 individually is K, and A, B, C, D, and
E are any amino acid, with the proviso, that the smallest distance
between a Y and a K is at least 5 amino acids.

59. The compound of claim 53 wherein the peptide of (I) is either a
fragment, consisting of at least seven consecutive amino acids of the FG
loop of the module of (i) or (ii), or a variant of such a fragment, and
the monomers of the oligomeric peptide of (II) each consist independently
of amino acid sequences of such fragments and variants.

60. A method of treating a disease or condition which comprises
administering to a subject suffering from said disease or condition a
therapeutically effective amount of at least one compound of claim 57,
said disease or condition being treatable with said compound.

61. The method according to claim 60, wherein the compound is used in
combination with a prosthetic device.

62. The method according to claim 60, wherein the administration is
continuous.

63. The method according to claim 61, wherein the device is a prosthetic
nerve guide.

64. The method of claim 60 which comprises treatment of a disease or
condition of the central or peripheral nervous system.

65. The method according to claim 60, which comprises treatment of a
disease or condition of the muscles; or treatment of a disease or
condition of the gonads, the pancreas, or the kidney.

66. The method according to claim 60, which prevents cell death of heart
muscle cells.

67. The method according to claim 66, which results in revascularisation.

68. The method according to claim 60, which promotes wound-healing.

69. The method according to claim 60, which inhibits angiogenesis.

70. The method according to claim 64, wherein the disease or condition is
a cancer.

71. The method according to claim 64, which results stimulation of the
ability to learn and/or of the short and/or long term memory.

73. A process of producing a pharmaceutical composition, comprising mixing
an effective amount of one or more of the compounds according to claim 1,
with one or more pharmaceutically acceptable additives or carriers.

74. A method of modulating an FGF receptor, in a cell, said FGF receptor
selected from the group consisting of FGF receptor I, FGF receptor II,
FGF receptor III and FGF receptor IV, which comprises introducing into
said cell a modulatory amount of the compound of claim 43.

75. The method of claim 74, wherein the modulation is of a human FGF
receptor in cells in a human.

76. The method of claim 74, wherein the human is suffering from a disease
or condition of the control or peripheral nervous system.

77. The method of claim 74, wherein the human is suffering from a disease
or condition of the gonads, the pancreas, or the kidney.

78. The method of claim 74, wherein the human is suffering from a wound.

79. The method of claim 74, wherein the human is suffering from cancer.

80. The method of claim 74, wherein the human is suffering from impaired
or subnormal ability to learn, short term memory, or long term memory.

Description:

[0001]The present invention relates to a compound comprising the third
Immunoglobulin (Ig3) module, and/or the fourth Immunoglobulin (Ig4)
module, and/or the fifth Immunoglobulin (Ig5) module, and/or the first
Fibronectin III (Fn3,1) module, and/or the second Fibronectin III (Fn3,2)
module of neural cell adhesion molecule (NCAM), or a fragment, or a
variant thereof, capable of interacting with an Fibroblast Growth Factor
(FGF) receptor and/or Adenosine-Tri-Phosphate (ATP) and/or L1, and
thereby the compounds are capable of inducing differentiation, modulating
proliferation, stimulate regeneration, neuronal plasticity and/or
survival of cells. Further, the present invention relates to a
pharmaceutical composition comprising said compound, a process of
producing a pharmaceutical composition and the use of said compound.

BACKGROUND OF THE INVENTION

[0002]Cell adhesion molecules (CAMs) constitute a group of proteins
mediating adhesion between cells. A major group of CAMs belongs to the
immunoglobulin (Ig) superfamily characterised by the presence of
immunoglobulin domains. The neural cell adhesion molecule (NCAM) is such
a cell adhesion molecule of the Ig superfamily that is particularly
abundant in the nervous system. NCAM is expressed on the external
membrane of nerve cells. When an NCAM molecule on one cell binds to
another NCAM molecule on another cell (homophilic binding), the binding
between the two cells is strengthened. NCAM not only binds to NCAM but
also to other proteins and/or glycoconjugates found on nerve cells or in
the extracellular matrix (heterophilic binding). NCAM also binds ATP.
NCAM interactions influence migration of cells, extension of neurites,
fasciculation of neurites, cell proliferation, cell survival, and
formation of synapses.

[0003]NCAM is encoded by a single gene, containing at least 25 exons. Due
to alternative splicing of precursor mRNA, a variety of mature mRNA
species and thereby protein isoforms of NCAM can be produced. Three major
NCAM isoforms are generated by alternative splicing of exons 15 and 18
determining the mode of attachment of NCAM to the plasma membrane and the
size of the intracellular NCAM domains, respectively. In the nervous
system a glycosylphosphatidyl inositol (GPI) anchored 120 kDa isoform is
expressed on the surface of glial cells, a transmembrane 140 kDa isoform
is expressed on both neurons and glial cells, whereas a transmembrane 180
kDa isoform is found predominantly on the surface of neurons. The
extracellular part of NCAM comprises five Ig-like homology modules (Ig1,
Ig2, Ig3, Ig4 and Ig5) and two fibronectin type III modules (F3,1 and
F3,2) (Berezin et al., 2000).

[0004]Heterophilic ligands of NCAM comprise a variety of heparan sulfate
proteoglycans (e.g. agrin) and chondroitin sulfate proteoglycans (e.g.
neurocan). NCAM Ig1 and Ig2 are probably the structural determinants of
the interaction of NCAM with heparan sulfate proteoglycans since these
two modules have been shown to bind heparin (Kiselyov et al. 1997).
Reports on whether the core protein or the carbohydrate moieties are
responsible for the binding of proteoglycans to NCAM are contradictory,
and the contribution of this interaction to NCAM-mediated cellular
functions is currently not understood (Retzler et al. 1996). The neural
cell adhesion molecule L1 and the fibroblast growth factor (FGF) receptor
are other heterophilic ligands of NCAM. The interaction between NCAM and
L1 has been shown to be mediated by N-linked oligo-mannosidic glycans
carried by L1 and a lectin-like binding site localised in the fourth Ig
module of NCAM. Through this binding NCAM has been suggested to
participate in a so-called assisted L1-L1 homophilic interaction
(Horstkorte et al., 1993) presenting an interesting example of
co-operation between two neural CAMs.

[0005]Three different models of homophilic binding have been suggested: 1)
a binding between the third Ig-like modules (Rao et al., 1992) of two
opposing molecules; 2) involvement of all five Ig-like modules in an
antiparallel interaction (Ranheim et al., 1996); and 3) a reciprocal
binding of the first and second Ig-like modules (Kiselyov et al., 1997).
The latter model has recently been confirmed by nuclear magnetic
resonance (NMR) analysis (Jensen et al., 1999) and X-ray crystallography
(Kasper et al., 2000).

[0006]NCAM plays a crucial role during the development of the nervous
system and of organs, such as kidney, bowel, heart, gonads, pancreas, and
muscles. In the mature nervous system NCAM is important for the
plasticity of neuronal connections associated with regeneration, learning
and memory. In the peripheral nervous system NCAM is involved in the
initiation of outgrowth of nerve fibres and formation of nerve-muscle
connections in regeneration after damage including lesions.

[0007]In signal transduction NCAM transduces extracellular signals leading
to tyrosine phosphorylation, such as for example of the FGF-receptor, and
an increase in intracellular calcium concentration.

[0009]NCAM binding compounds capable of stimulating differentiation and/or
neurite outgrowth from cells presenting NCAM are disclosed in WO
00/18801, in which the compounds are used in the treatment for
regeneration of NCAM presenting cells.

[0010]The identification of one such compound, C3, is described by Ronn et
al. (1999). C3 stimulates outgrowth by activating a signalling pathway
identical to that activated by homophilic NCAM binding, but it does not
bind directly to FGF receptors.

[0011]Various factors may cause neuronal cell death. Preventing neuronal
cell death in individuals being exposed to risk factors causing cell
death may be called maintaining/stimulating or promoting survival of the
cells, or it may be called neuroprotection.

[0012]When neuronal cells are damaged, e.g. by reduced oxygen supply, the
processes of cell death start and lead to cellular dysfunction,
"collapse" of the intercellular communication between cells (network),
retraction of cell processes and eventually cell death. Preventing
neuronal cell death, i.e. stimulating/promoting survival means that the
cells are protected from initiation of the processes of cell death.

[0013]Survival of nerve cells has been discussed in some references, for
example Hulley et al. (1998) disclose that the L1 neural cell adhesion
molecule is capable of stimulating survival and differentiation in fetal
mid-brain dopaminergic neurons cultured in the presence of the toxin
MPP+.

[0014]U.S. Pat. No. 6,037,320 describes the identification of a
neurotrophic factor, NT-4 and in U.S. Pat. No. 5,767,240 an
activity-dependent neurotrophic factor capable of increasing the survival
of spinal cord neuronal cells, cerebral cortical cells and hippocampal
neurons is revealed.

[0015]Further, U.S. Pat. No. 5,567,682 concerns a method of treating the
symptoms of Alzheimer's disease by intranasal administration of short
chain peptides. The peptides promote neuronal survival by reducing or
halting progressive neuronal degeneration.

[0016]NCAM has recently been demonstrated to have an ecto-adenosine
triphosphatase (ATPase) activity (Dzhandzhugazyan and Bock, 1993 and
1997). The role of this activity in ATP is one of the most abundant
neurotransmitters in the nervous system. In a recent study it has been
demonstrated that ATP modulates NCAM induced neurite outgrowth,
indicating that ATP may be a regulator of the putative NCAM-FGF-receptor
signalling pathway (Skladchikova et al., 1999).

[0017]However, the inventors of the present invention have surprisingly
found that a compound comprising the third Immunoglobulin (Ig3) module,
and/or the fourth immunoglobulin (Ig4) module, and/or the fifth
Immunoglobulin (Ig5) module, and/or the first Fibronectin III (Fn3,1)
module, and/or the second Fibronectin III (Fn3,2) module of neural cell
adhesion molecule (NCAM), or a fragment, or a variant thereof is capable
of inducing differentiation, modulating proliferation, stimulating
regeneration, neuronal plasticity and survival of cells through an
interaction with the Fibroblast Growth Factor (FGF) receptor and/or
adenosine-tri-phosphate (ATP) and/or L1.

SUMMARY OF THE INVENTION

[0018]The present invention concerns a compound comprising the third
Immunoglobulin (Ig3) module, and/or the fourth Immunoglobulin (Ig4)
module, and/or the fifth Immunoglobulin (Ig5) module, and/or the first
Fibronectin III (Fn3,1) module, and/or the second Fibronectin III (Fn3,2)
module of neural cell adhesion molecule (NCAM), or a fragment, or a
variant thereof, capable of interacting with Fibroblast Growth Factor
(FGF) receptor and/or Adenosine-Tri-Phosphate (ATP) and/or L1. In the
present context first Fibronectin III module and second Fibronectin III
module are equal to the denomination "F3,1 and F3,2" or "FnIII,1 and
FnIII,2" or "Fn3,1 and Fn3,2".

[0019]In a further aspect the invention concerns a compound comprising the
fourth Immunoglobulin (Ig4) module, and/or the fifth Immunoglobulin (Ig5)
module, and/or the first Fibronectin III (Fn3,1) module, and/or the
second Fibronectin III (Fn3,2) module of neural cell adhesion molecule
(NCAM), or a fragment, or a variant, capable of interacting with the FGF
receptor and/or adenosine-tri-phosphate (ATP) and/or L1.

[0021]Further, the invention describes a pharmaceutical composition
comprising at least one compound of the invention, and a process of
producing such a pharmaceutical composition. Also, the use of a compound
of the invention is within the scope of the invention as well as a method
for treating diseases and conditions with the compound.

FIGURES

[0022]FIG. 1. shows the structure of NCAM F3,2 determined by means of
1H,15N NMR spectros copy. Structure of the second F3 module of
NCAM, a) Stereo view of an overlay of the backbone atoms of 30
superimposed structures, b) Ribbon representation of the structure. The
structure consists of 7 anti-parallel β-strands arranged in a
sandwich of two β-sheets, one containing three strands (ABE) and the
other four strands (CDFG).

[0023]FIG. 2. Demonstration of interaction between the second F3 module of
NCAM and the third Ig module of the FGF-receptor or ATP. a-d) Changes in
the chemical shifts of 1H and 15N atoms of 0.05 mM 15N
labeled sample of the second F3 module of NCAM after addition of 1 mM
unlabeled sample of the third Ig module of the FGF-receptor (a, b) or 5
mM AMP-PCP (c, d). e) Mapping of the residues of the second F3 module of
NCAM with changes in the chemical shifts (in the presence of the third Ig
module of the FGF-receptor) greater than 0.006 ppm for 1H or 0.03
ppm for 15N atoms, onto the structure of the module. The residues
with strong changes in the chemical shifts (greater than 0.01 ppm for
1H or 0.1 ppm for 15N atoms) are colored blue (shown by A) and
with weak changes--red (shown by B); all other residues are colored
yellow (shown by C). f) Mapping of the residues of the second F3 module
perturbed by AMP-PCP (blue color--shown by C) and the residues of the ATP
binding Walker motif A (red color--shown by A) and Lys 85 (green
color--shown by B) to the structure of the module; all other residues are
colored yellow (shown by D). g) A possible arrangement of the complex of
the second F3 module of NCAM with ATP.

[0024]FIG. 3. Effect of the second F3 module, its FGF-receptor binding
part (the FG loop peptide) on phosphorylation of the FGF-receptor 1.
HEK293 cells, transiently transfected with a His-tagged version of the
FGF-receptor 1, were stimulated for 20 min with either 5 μg/ml F3,2
module or 50 μg/ml FG loop peptide. a) The total amount of the
FGF-receptor 1 and the amount of the FGF-receptor phosphorylation was
estimated by immunoblotting using anti-pentahis (anti-His) and
anti-phosphotyrosine (anti-P-tyr) antibodies, respectively. b)
Quantification of the FGF-receptor phosphorylation by densitometric
analysis of the band intensity. Phosphorylation was estimated relative to
the control (untreated cells), which has been normalized to 1.0. Error
bar represents one standard deviation (SD). P<0.05 by paired t test
comparing treated cells with controls. The t test was performed on array
of six independent sets of non-normalized data.

[0025]FIG. 4. Effect of the second F3 module and its FGF-receptor binding
part (the FG loop peptide) on neurite outgrowth from hippocampal neurons.
a) Micrographs of the control (untreated) neurons. b) Micrographs of
neurons treated with 5 μM second F3 module. c) Plot of the neurite
length versus the concentration of the second F3 module, the FG-loop
peptide and a truncated version of the peptide. d) Effect of an
anti-FGF-receptor antibody on neurite outgrowth induced by 5 μM second
F3 module or 50 μM FG loop peptide.

[0026]FIG. 5. Effect of various modifications of the FG loop peptide and a
peptide derived from basic FGF on their peptides potency to stimulate
neurite outgrowth from hippocampal neurons. Concentration of the various
peptides was 50 μM. a) Effect of truncations and substitutions of the
various amino acids with Ala in the FG loop peptide or truncated versions
of the peptide. b) effect of substitution of the various amino acids with
Ala in a peptide derived from basic FGF.

[0027]FIG. 6. Sequential and structural similarity between heptameric
peptides derived from the FGF-receptor binding part of NCAM and basic
FGF. a) Sequential alignment of the heptamers, in which signs "|", " ",
":", and "•" indicate the level of similarity in a decreasing order
from strong to low similarity. b) Structural alignment of the backbone
atoms of the heptamers from NCAM (blue color--shown by A) and basic FGF
(red color--shown by B).

[0028]FIG. 7. Effect of ATP (a) and AMP-PCP (b) on the potency of the
second F3 module, the FG loop peptide and a modified version of the
peptide to stimulate neurite outgrowth from hippocampal neurons. Neurons
were stimulated with either 5 μM second F3 module or 50 μM peptide
in the presence of various concentrations of ATP or AMP-PCP (0, 0.4, 1.0
mM). "cnt" stands for control, "F3"--the second F3 module, "FGL"--the FG
loop peptide, and "YKK"--the FGL peptide in which Tyr 74, Lys 83 and Lys
85 were substituted for Ala.

[0029]FIG. 8. Cerebellar granule neurons from seven days old rats were
grown in the presence of 40 mM potassium. The cells were subsequently
transferred to serum-free medium containing only five mM potassium and
grown for two days in serum-free medium supplemented with various
concentrations of he FGL-peptide (see FIG. 8). Subsequently, the number
of cells were determined and the amount of cells surviving in the
presence of high-concentration of potassium was set at 100%. As can be
seen approx. Only 60% survived in the presence of brains-derived
neurotrophic factor or basic fibroblast growth factor. When FGL was added
in a sode-range of 2-250 microgram per ml statistically significant
survival was observed up to 90% of the positive control at a dose of 250
microgram per ml of the monomeric form of the FGL peptide.

[0030]FIG. 9a. Peptides derived from the FG-lops of the neural cell
adhesion molecules L1 and NCAM (third F3-module of L1 and first F3 module
of NCAM) were prepared in different lengths, see FIG. 9a, and their
effect on neurite outgrowth from primary hippocampal neurons were tested
adding the various peptides in a concentration of 25 microM. FIG. 9b. The
NCAM peptides are referred to as FN3,1 and the L1 peptides are referred
to as L1. The variants indicated in FIG. 9a are indicated by the number
of amino acids in each peptide. As can be seen from the figure, the
peptides had a stimulatory effect on neurite outgrowth reaching
statistically significance for the nine amino acid variant of the Fgloop
of first fibronectin type III module of NCAM and the nine amino acid
variant of the FG-loop of the third fibronectin type III-module of L1.

DETAILED DESCRIPTION OF THE INVENTION

[0031]The compound according to the invention relates to the induction of
differentiation, modulation of proliferation, stimulation of
regeneration, neuronal plasticity and survival of cells.

[0032]By the term FGL peptide is meant FG loop peptide of NCAM, which is
an FGF receptor binding site of NCAM corresponding to SEQ ID NO: 1.

[0033]By the term "modulation" is meant a change, for example either an
inhibition or a stimulation.

[0034]In the present context the term "interacting" refers to the direct
or indirect contact between a compound of the invention and the FGF
receptor, preferably a direct interaction. The term "direct interaction"
means that the compound in question binds directly to the receptor.

[0035]By the term "cells presenting the FGF receptor" is meant cells
expressing the FGF receptor on the external membrane of the cells, these
cells are for example neurons, glial cells, all types of muscle cells,
neuroendocrine cells, gonadal cells and kidney cells, endothelial cells
and fibroblasts.

[0036]By the term "cells presenting an NCAM ligand" is meant cells
expressing a receptor or ligand whereto NCAM and/or parts of NCAM may
bind (i.e.: so-called counter-receptor). Examples of NCAM ligands are the
FGF (fibroblast growth factor) receptor, L1 or glyco-conjugates or
glucose-aminoglycans, such as heparin, heparan sulphateproteoglycans, and
chondroitin sulphate proteoglycans and ATP.

[0037]In the present context the wording "stimulate/promote survival" is
used synonymously with the wording "preventing cell death" or
"neuroprotection". By stimulating/promoting survival it is possible to
prevent diseases or prevent further degeneration of the nervous system in
individuals suffering from a degenerative disorder.

[0038]"Survival" refers to the process, wherein a cell has been
traumatised and would under normal circumstances, with a high probability
die, if not the compound of the invention was used to prevent said cell
from degenerating, and thus promoting or stimulating survival of said
traumatised cell.

[0039]"Neuronal plasticity" refers to the capability of remodelling
neuronal connections.

[0040]Peripheral nerve cells possess to a limited extent a potential to
regenerate and re-establish functional connections with their targets
after various injuries. However, functional recovery is rarely complete
and peripheral nerve cell damage remains a considerable problem. In the
central nervous system, the potential for regeneration is even more
limited. Therefore, the identification of substances with the ability to
prevent neuronal cell death in the peripheral and the central nervous
system is significant and of great commercial value.

[0041]Accordingly, the present invention relates to the finding that a
compound comprising the third Immunoglobulin (Ig3) module, and/or the
fourth Immunoglobulin (Ig4) module, and/or the fifth Immunoglobulin (Ig5)
module, and/or the first Fibronectin III (F3,1) module, and/or the second
Fibronectin III (F3,2) module of neural cell adhesion molecule (NCAM), or
a fragment, or a variant, is capable of interacting with the Fibroblast
Growth Factor (FGF) receptor and or Adenosine-Tri-Phosphate (ATP) and/or
L1.

[0042]In the present context the NCAM molecule referred to is NCAM having
the sequence shown in database SWISSPROT, accession No: P13591

[0043]In this sequence the position of the domains mentioned herein are as
follows:

[0044]Further the invention concerns the finding that a compound
comprising the fourth Immunoglobulin (Ig4) module, and/or the fifth
Immunoglobulin (Ig5) module, and/or the first Fibronectin III (Fn3,1)
module, and/or the second Fibronectin III (Fn3,2) module of neural cell
adhesion molecule (NCAM), or a fragment, or a variant thereof, is capable
of interacting with the FGF receptor and/or Adenosine-Tri-Phosphate (ATP)
and/or L1, such as a compound comprising the fourth Immunoglobulin (Ig4)
module, and/or the first Fibronectin III (Fn3,1) module, and/or the
second Fibronectin III (Fn3,2) module of neural cell adhesion molecule
(NCAM), or a fragment, or a variant thereof, is capable of interacting
with the FGF receptor and/or Adenosine-Tri-Phosphate (ATP) and/or L1, or
such as a compound comprising the fifth Immunoglobulin (Ig5) module,
and/or the first Fibronectin III (Fn3,1) module, and/or the second
Fibronectin III (Fn3,2) module of neural cell adhesion molecule (NCAM),
or a fragment, or a variant thereof, is capable of interacting with the
FGF receptor and/or Adenosine-Tri-Phosphate (ATP) and/or L1, such as a
compound comprising the first Fibronectin III (Fn3,1) module, and/or the
second Fibronectin III (Fn3,2) module of neural cell adhesion molecule
(NCAM), or a fragment, or a variant thereof, is capable of interacting
with the FGF receptor and/or Adenosine-Tri-Phosphate (ATP) and/or L1.

[0045]In the present context the "fragment thereof" is to be understood as
being any part of the NCAM molecule capable of interacting with an
FGF-receptor and/or ATP and/or L1 and through said binding modulate
proliferation, and/or induce differentiation, and/or stimulate
regeneration, neuronal plasticity and/or survival of cells. The "variant
thereof" is to be understood as being any peptide sequence capable of
interacting with FGF-receptors and/or ATP and/or L1, and via said binding
induce differentiation, modulate proliferation, stimulate regeneration,
neuronal plasticity and survival of cells. Thus, fragment or variant may
be defined as [0046]i) Fragments/variants comprising an amino acid
sequence capable of being recognised by an antibody also capable of
recognising the predetermined NCAM amino acid sequence, and/or [0047]ii)
Fragments/variants comprising an amino acid sequence capable of binding
to a receptor moiety also capable of binding the predetermined NCAM amino
acid sequence, and/or [0048]iii) Fragments/variants having at least a
substantially similar binding affinity to at least one FGF receptor
and/or ATP and/or L1 as said predetermined NCAM amino acid sequence.

[0049]In the present context the term "functional equivalent" means a
variant as defined above.

[0050]The binding affinity of the compound according to the invention
preferably has a binding affinity (Kd value) to NCAM and/or the NCAM
ligand in the range of 10-3 to 10-10 M, such as preferably in
the range of 10-4 to 10-8 M. According to the present invention
the binding affinity is determined by one of the following assays of
surface plasmon resonance analysis or nuclear magnetic resonance
spectroscopy.

[0051]In the present context, a variant of the NCAM domains mentioned
above is to be understood as being any compound interacting with any cell
presenting a FGF receptor or an NCAM ligand, and/or L1 and through said
interaction modulates proliferation, and/or induce differentiation,
and/or stimulate regeneration, neuronal plasticity and/or survival of FGF
receptor presenting cells, i.e. functional variants. Variants may be
peptides, peptide derivatives, antibodies and non-peptide compounds such
as small organic compounds, sugars and fats, as well as peptido-mimetics.
In a preferred embodiment the variant is a peptide as discussed above.

[0052]In one embodiment variants may be understood to exhibit amino acid
sequences gradually differing from the preferred predetermined sequence,
as the number and scope of insertions, deletions and substitutions
including conservative substitutions increase. This difference is
measured as a reduction in homology between the predetermined sequence
and the variant.

[0053]The peptides may be modified, for example by substitution of one or
more of the amino acid residues. Both L-amino acids and D-amino acids may
be used. Other modification may comprise derivatives such as esters,
sugars, etc. Examples are methyl and acetyl esters. Polymerisation such
as repetitive sequences or attachment to various carriers are well-known
in the art, e.g. lysine backbones, such as lysine dendrimers carrying 4
peptides, 8 peptides, 16 peptides, or 32 peptides. Other carriers may be
protein moieties, such as bovine serum albumin (BSA), or lipophilic
dendrimers, or micelle-like carriers formed by lipophilic derivatives, or
starburst (star-like) carbon chain polymer conjugates.

[0054]Variants of the fragments according to the invention may comprise,
within the same variant, or fragments thereof or among different
variants, or fragments thereof, at least one substitution, such as a
plurality of substitutions introduced independently of one another.
Variants of the complex, or fragments thereof may thus comprise
conservative substitutions independently of one another, wherein at least
one glycine (Gly) of said variant, or fragments thereof is substituted
with an amino acid selected from the group of amino acids consisting of
Ala, Val, Leu, and Ile, and independently thereof, variants, or fragments
thereof, wherein at least one alanine (Ala) of said variants, or
fragments thereof is substituted with an amino acid selected from the
group of amino acids consisting of Gly, Val, Leu, and Ile, and
independently thereof, variants, or fragments thereof, wherein at least
one valine (Val) of said variant, or fragments thereof is substituted
with an amino acid selected from the group of amino acids consisting of
Gly, Ala, Leu, and Ile, and independently thereof, variants, or fragments
thereof, wherein at least one leucine (Leu) of said variant, or fragments
thereof is substituted with an amino acid selected from the group of
amino acids consisting of Gly, Ala, Val, and Ile, and independently
thereof, variants, or fragments thereof, wherein at least one isoleucine
(Ile) of said variants, or fragments thereof is substituted with an amino
acid selected from the group of amino acids consisting of Gly, Ala, Val
and Leu, and independently thereof, variants, or fragments thereof
wherein at least one aspartic acids (Asp) of said variant, or fragments
thereof is substituted with an amino acid selected from the group of
amino acids consisting of Glu, Asn, and Gln, and independently thereof,
variants, or fragments thereof, wherein at least one aspargine (Asn) of
said variants, or fragments thereof is substituted with an amino acid
selected from the group of amino acids consisting of Asp, Glu, and Gln,
and independently thereof, variants, or fragments thereof, wherein at
least one glutamine (Gln) of said variants, or fragments thereof is
substituted with an amino acid selected from the group of amino acids
consisting of Asp, Glu, and Asn, and wherein at least one phenylalanine
(Phe) of said variants, or fragments thereof is substituted with an amino
acid selected from the group of amino acids consisting of Tyr, Trp, His,
Pro, and preferably selected from the group of amino acids consisting of
Tyr and Trp, and independently thereof, variants, or fragments thereof,
wherein at least one tyrosine (Tyr) of said variants, or fragments
thereof is substituted with an amino acid selected from the group of
amino acids consisting of Phe, Trp, His, Pro, preferably an amino acid
selected from the group of amino acids consisting of Phe and Trp, and
independently thereof, variants, or fragments thereof, wherein at least
one arginine (Arg) of said fragment is substituted with an amino acid
selected from the group of amino acids consisting of Lys and His, and
independently thereof, variants, or fragments thereof, wherein at least
one lysine (Lys) of said variants, or fragments thereof is substituted
with an amino acid selected from the group of amino acids consisting of
Arg and His, and independently thereof, variants, or fragments thereof,
and independently thereof, variants, or fragments thereof, and wherein at
least one proline (Pro) of said variants, or fragments thereof is
substituted with an amino acid selected from the group of amino acids
consisting of Phe, Tyr, Trp, and His, and independently thereof,
variants, or fragments thereof, wherein at least one cysteine (Cys) of
said variants, or fragments thereof is substituted with an amino acid
selected from the group of amino acids consisting of Asp, Glu, Lys, Arg,
His, Asn, Gln, Ser, Thr, and Tyr.

[0055]It is clear from the above outline that the same equivalent or
fragment thereof may comprise more than one conservative amino acid
substitution from more than one group of conservative amino acids as
defined herein above.

[0056]Conservative substitutions may be introduced in any position of a
preferred predetermined peptide of the invention or fragment thereof. It
may however also be desirable to introduce non-conservative
substitutions, particularly, but not limited to, a non-conservative
substitution in any one or more positions.

[0057]A non-conservative substitution leading to the formation of a
functionally equivalent fragment of the peptide of the invention would
for example differ substantially in polarity, for example a residue with
a non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met)
substituted for a residue with a polar side chain such as Gly, Ser, Thr,
Cys, Tyr, Asn, or Gln or a charged amino acid such as Asp, Glu, Arg, or
Lys, or substituting a charged or a polar residue for a non-polar one;
and/or ii) differ substantially in its effect on peptide backbone
orientation such as substitution of or for Pro or Gly by another residue;
and/or iii) differ substantially in electric charge, for example
substitution of a negatively charged residue such as Glu or Asp for a
positively charged residue such as Lys, His or Arg (and vice versa);
and/or iv) differ substantially in steric bulk, for example substitution
of a bulky residue such as His, Trp, Phe or Tyr for one having a minor
side chain, e.g. Ala, Gly or Ser (and vice versa).

[0058]Substitution of amino acids may in one embodiment be made based upon
their hydrophobicity and hydrophilicity values and the relative
similarity of the amino acid side-chain substituents, including charge,
size, and the like. Exemplary amino acid substitutions which take various
of the foregoing characteristics into consideration are well known to
those of skill in the art and include: arginine and lysine; glutamate and
aspartate; serine and threonine; glutamine and asparagine; and valine,
leucine and isoleucine.

[0059]The addition or deletion of an amino acid may be an addition or
deletion of from 2 to preferably 10 amino acids, such as from 2 to 8
amino acids, for example from 2 to 6 amino acids, such as from 2 to 4
amino acids. However, additions of more than 10 amino acids, such as
additions from 2 to 10 amino acids, are also comprised within the present
invention. In the multimeric forms additions/deletions may be made
individually in each monomer of the multimer.

[0061]It will thus be understood that the invention concerns a compound
comprising at least one fragment capable of binding at least one
receptor, or a variant thereof including any variants and functional
equivalents of such at least one fragment.

[0062]A functional equivalent obtained by substitution may well exhibit
some form or degree of native NCAM activity, and yet be less homologous,
if residues containing functionally similar amino acid side chains are
substituted. Functionally similar in the present respect refers to
dominant characteristics of the side chains such as hydrophobic, basic,
neutral or acidic, or the presence or absence of steric bulk.
Accordingly, in one embodiment of the invention, the degree of identity
between i) a given functional equivalent capable of effect and ii) a
preferred predetermined fragment, is not a principal measure of the
fragment as a variant or functional equivalent of a preferred
predetermined peptide fragment according to the present invention.

[0063]Fragments sharing at least some homology with a preferred
predetermined fragment of at least 3 amino acids, more preferably at
least 5 amino acids, are to be considered as falling within the scope of
the present invention when they are at least about 25 percent homologous
with the preferred predetermined NCAM peptide, or fragment thereof, such
as at least about 30 percent homologous, for example at least about 40
percent homologous, such as at least about 50 percent homologous, for
example at least about 55 percent homologous, such as at least about 60
percent homologous, for example at least about 65 percent homologous,
such as at least about 70 percent homologous, such as at least about 75
percent homologous, for example at least about 80 percent homologous,
such as at least about 85 percent homologous.

[0064]Sequence identity can be measured using sequence analysis software
(for example, the Sequence Analysis Software Package of the Genetics
Computer Group, University of Wisconsin Biotechnology Centre, 1710
University Avenue, Madison, Wis. 53705), with the default parameters as
specified therein.

[0065]Throughout the description and claims either the three letter code
or the one letter code for natural amino acids are used. Where the L or D
form has not been specified it is to be understood that the amino acid in
question has the natural L form, cf. Pure & Appl. Chem. Vol. (56(5) pp
595-624 (1984) or the D form, so that the peptides formed may be
constituted of amino acids of L form, D form, or a sequence of mixed L
forms and D forms.

[0066]Where nothing is specified it is to be understood that the
C-terminal amino acid of a polypeptide of the invention exists as the
free carboxylic acid, this may also be specified as "--OH". However, the
C-terminal amino acid of a compound of the invention may be the amidated
derivative, which is indicated as "--NH2". Where nothing else is
stated the N-terminal amino acid of a polypeptide comprise a free
amino-group, this may also be specified as "H--".

[0068]In one aspect of the invention the compound comprises the first
Fibronectin III (F3,1) module of NCAM, or a fragment, or a variant
thereof.

[0069]More particularly the invention relates to a compound comprising the
first Fibronectin III (F3,1) module of neural cell adhesion molecule
(NCAM), or a fragment, or a variant thereof capable of interacting with
the FGF receptor.

[0070]Thus, in one preferred embodiment of the invention the compound
comprises an amino acid sequence of the formula

L1-A-L2-B-L3-C-L4 [0071]wherein [0072]one of A, B, C is selected from a
basic amino acid, [0073]one of A, B, C is selected from a hydrophobic
amino acid, [0074]one of A, B, C is glycine, and [0075]L1, L2, L3, L4 may
be selected from a chemical bond or an amino acid sequence having n amino
acid residues, wherein n is an integer of from 0 to 5.

[0076]In yet another embodiment B is glycine, A is a basic amino acid
residue, and C is a hydrophobic amino acid residue.

[0077]Further, in another embodiment A is lysine (K) or arginine (R), and
C is leucine (L) or alanine (A).

[0078]In another embodiment the compound of the invention comprises the
sequence NGKGL (Aspargine, Glycine, Lysine, Glycine, Leucine), NGKGA,
NGRGL and/or NGRGA. The sequence NGRGL is for example found in L1 and the
sequence NGKGL is for example found in F3,1 of NCAM.

[0079]The invention further discloses that F3,2 is a ligand of the FGF
receptor and is capable of interacting with cells presenting the FGF
receptor. Thus, in another aspect of the invention the compound comprises
a peptide homologous to the FG loop of the second FnIII module of the
NCAM molecule, or a fragment, or a variant thereof.

[0080]A fragment comprising the FG loop of the F3,2 module of NCAM is
particularly preferred. However, the invention is not limited to
fragments of the F3,2 module comprising the FG loop. Deletions of such
fragments generating functionally equivalent fragments comprising less
than the FG loop are also comprised within the present invention.
Functionally equivalent peptides and fragments thereof according to the
present invention, may comprise less or more amino acid residues than the
FG loop of the F3,2 module capable of binding to cells presenting an FGF
receptor and/or NCAM ligands.

[0081]All functional equivalents of F3,2 peptides are included within the
scope of this invention, regardless of the degree of homology that they
show to a predetermined sequence of the F3,2 peptide or FG loop. The
reason for this is that some parts of the binding regions are most likely
readily mutatable, or capable of being peptide deleted, without any
significant effect on the binding activity of the resulting fragment.

[0082]Such a peptide, fragment or variant may be a compound comprising an
amino acid sequence of the formula

L1-A-L2-B-L3-C-L4-D-L5 [0083]wherein [0084]one of A, B, C, D is selected
from a basic amino acid residue, [0085]one of A, B, C, D is selected from
a hydrophobic amino acid residue, [0086]one of A, B, C, D is selected
from an acidic amino acid residue, [0087]one of A, B, C, D is glycine,
and [0088]L1, L2, L3, L4 and L5 may be selected from a chemical bond or
an amino acid sequence having n amino acid residues, wherein n is an
integer of from 0 to 5.

[0089]In particular a peptide, fragment or variant may be a compound
comprising an amino acid sequence of the formula

A-B-L3-L4-C-L4 wherein [0090]A is a hydrophobic amino acid residue,
[0091]B is an acidic amino acid residue, [0092]L3 is one or more
hydrophilic amino acid residue(s), [0093]L4 is an amino acid sequence as
defined above for L4, and [0094]C is glycine.

[0095]In a preferred embodiment the compound comprise an amino acid
sequence with the formula

AENQ-L4-G, [0096]wherein A, E, N, Q, and G are the one-letter notation
for amino acid residues, and L4 may be selected from a chemical bond or
an amino acid sequence having n amino acid residues, wherein n is an
integer of from 0 to 5.

[0097]In another embodiment a peptide, fragment or variant according to
the invention may comprise a peptide homologous to the FG loop of the
Fn3,2 module of the NCAM molecule, or a fragment, or a variant thereof.
In one embodiment of the invention the peptide comprises a sequence
having the amino acid residue motif A-E-N-Q-X-X-K, wherein X may be any
amino acid residue. X may for example be selected individually from
Glutamine (Q), Alanine (A), Glycine (G) and/or Asparagine (N).

[0098]In particular a peptide, fragment or variant may be a compound
comprising an amino acid sequence of the formula

A-L2-B-L3-L4-C-L4 wherein [0099]A is a hydrophobic amino acid residue,
[0100]L2 may be selected from a chemical bond or an amino acid sequence
having n amino acid residues, wherein n is an integer of from 0 to 5
[0101]B is a basic amino acid residue, [0102]L3 is one or more
hydrophilic amino acid residue(s), [0103]L4 may be selected from a
chemical bond or an amino acid sequence having n amino acid residues,
wherein n is an integer of from 0 to 5, and [0104]C is glycine.

[0105]In a preferred embodiment the compound comprise an amino acid
sequence with the formula

AM-B-L3-L4-G, [0106]wherein A, M, and G are the one-letter notation for
amino acid residues, L3 is one or more hydrophilic amino acid residue(s),
and L4 may be selected from a chemical bond or an amino acid sequence
having n amino acid residues, wherein n is an integer of from 0 to 5.

[0107]Examples may be the following sequence: AMKEDGR (SEQ ID NO: 7)

[0108]In yet another aspect of the invention the compound is capable of
binding adenosine-tri-phosphate (ATP). Without being bound by theory it
is speculated that release of ATP from the synapse may effect the
coupling between NCAM and the FGF-receptor and therefore regulate the
axonal growth in the area of a newly formed synaptic contact, i.e. that
ATP indirectly effects the plasticity in the area of synaptic contact.

[0109]In one such embodiment the compound comprises a sequence of the
formula

L1-A-L2-B-L3-C-L4-D-L5-E-L6, wherein

at least one of L1, L2, L3, or L4 comprises the amino acid residue Y and
one of the other comprises the amino acid residue K, and L5 and/or L6
individually is K, and A, B, C, D, E is any amino acid, with the proviso,
that the distance between Y and K is at least 5 amino acids, such as at
least 7 amino acid residues, such as at least 9 amino acid residues, such
as at least 11 amino acid residues.

[0110]It is preferred that the amino acid Y is in closer proximity to the
N-terminal than the amino acid K.

[0111]In a further embodiment of the invention the compound comprises a
sequence of the formula

A-Xaa-B-C-C, wherein [0112]A is tyrosine (Y), [0113]B is glycine (G),
[0114]C is lysine (K), and [0115]Xaa is any amino acid

[0116]In a preferred embodiment the compound according to the invention
comprises at least one peptide comprising the sequence

[0117]The sequences listed above may be part of naturally occurring
proteins, for example the peptide having the sequence of AMKEDGR (SEQ ID
NO 7) is found in the Fibroblast Growth factor 2 (FGF 2).

[0118]In one embodiment of the invention the sequences 86-206 are
homologue sequences to the

[0119]FGL peptide (FG loop peptide) in the F3,1 and F3,11 domains.

[0120]By the term "homologue" is meant a sequence which is structurally
and/or functionally identical with the FGL peptide of the invention.
Sequence identity can be measured using sequence analysis software (for
example, the Sequence Analysis Software Package of the Genetics Computer
Group, University of Wisconsin Biotechnology Center, 1710 University
Avenue, Madison, Wis. 53705), with the default parameters as specified
therein.

[0121]In one embodiment of the invention the compound is capable of
interacting with the Fibroblast Growth Factor (FGF) receptor. A variety
of FGF receptors exist. It is preferred that the FGF receptor may be
selected from FGF receptor I, FGF receptor II, FGF receptor III, FGF
receptor IV. In a more preferred embodiment the FGF receptor I signalling
is stimulated.

[0122]In a preferred embodiment of the invention the interaction of the
present compound with the FGF receptor is resulting in the stimulation of
FGF receptor signalling. When the compound of the invention interacts
with the FGF receptor a cascade of chemical events and physiological
changes occurs. The interaction of the present compound with the FGF
receptor causes presumably conformational changes or clustering of the
receptor by which chemical signals are created and propagated from the
site of interaction to the inside of the cell. The signals are said to be
transduced from the outside to the inside of the cell, the latter
resulting in a physiological response of the cell.

[0123]According to the invention the FGF receptor signalling is measured
as phosphorylation of the FGF receptor when a predetermined concentration
of the compound is applied to cells expressing the FGF receptor. The
degree of phoshorylation is at least 20% above the control value, such as
at least 20-200%, for example at least 50-200%.

[0124]When testing the present compound with respect to for example
measuring signalling the concentration of the said compound may be
between 0.1-1000 μM, 1-1000 μM, for example 1-200 μM, for
example 10-200 μM, such as 20-180 μM, for example 30-160 μM,
such as 4-140 μM, for example 50-130 μM, such as 60-120 μM, for
example 70-110 μM, such as 80-100 μM.

[0125]The amino acid sequence of the compound of the invention may be of
any suitable length, in that the length of the amino acid sequence is
dictated by the functionality of the peptide and the formulation of the
compound into a pharmaceutical composition. Thus, the compound normally
comprises amino acid residues in the range of from 3-100 amino acid
residues, such as from 10-90 amino acid residues, for example from 15-85
amino acid residues, such as from 20-80 amino acid residues, for example
from 25-75 amino acid residues, such as from 30-70 amino acid residues,
for example from 35-65 amino acid residues, such as from 40-60 amino acid
residues, for example from 45-55 amino acid residues.

[0126]In another aspect the compound comprises amino acid residues in the
range of from 3 to 20 amino acid residues, such as from 3-19 amino acid
residues, for example from 3-18 amino acid residues, such as from 3-17
amino acid residues, for example from 3-16 amino acid residues, such as
from 3-15 amino acid residues, for example from 3-14 amino acid residues,
such as from 3-13 amino acid residues.

[0127]The peptides of the invention may serve as tools for identifying a
motif in peptide ligands expected to bind to the FGF receptor and/or ATP.
Such peptide ligands may be found through a peptide and/or a non-peptide
library. Any peptide sequence comprising said peptides capable of binding
the FGF receptor and/or ATP and/or L1 are part of the present invention.

[0128]These mentioned compounds and compositions can be used to treat
conditions affecting the peripheral and/or the central nervous system
and/or muscles and other tissues expressing FGF receptors or NCAM ligands
as well as other conditions in which a stimulation of FGF receptor
function or the function of other NCAM ligand is beneficial.

[0129]Putative artificial ligands may be selected and identified from
peptide or non-peptide libraries. Any peptide or non-peptide library may
be used. Synthetic peptide and non-peptide libraries as well as libraries
containing fragmented natural occurring proteins, may be used in the
search for useful peptides. Any kind of libraries comprising non-peptide
compounds may similarly be used.

[0130]Peptides characterised by a certain sequence of amino acids may be a
variant of a certain area of a protein. Naturally occurring proteins
consist of L-amino acid residues. However, artificial peptides may also
consist of or comprise D-amino acid residues. By combinatorial chemistry,
mixtures of beads carrying peptides of equal length can be constructed,
in which each bead carries peptides of a unique sequence (Lam et al.,
1991). Such a mixture of peptides on beads is called a peptide library.

[0131]In the present invention, peptides, fragments or variants may be
identified by screening synthetic random peptide libraries comprising
resin-bound peptides with purified recombinant NCAM or recombinant FGF
receptor or recombinant L1 or other NCAM ligands. The synthesis of the
resin-bound one-bead one-peptide library may be performed using the
portioning, mix procedure of Furka, ., Sebestyyen, F., Asgedom, M. And
Dibo, G. (1991) Int. J. Pep. Prot. Res. 37, 487-493) optionally modified
as known to the person skilled in the art. It is to be understood that
the method chosen for identification and selection of interesting
peptides is not critical for the identification of a putative motif.

[0132]Libraries of small organic compounds may be screened to identify FGF
receptor ligands or L1 ligands or other NCAM counter-receptor ligands
capable of interacting with Fibroblast Growth Factor (FGF) receptor
and/or Adenosine-Tri-Phosphate (ATP) and/or L1. Such libraries or their
construction are commonly known and the screening for useful ligands may
follow the methods for screening disclosed in the present specification,
or in ways obvious to the skilled person.

[0133]The compound of the present invention may preferably be in the form
of an oligomer (multimer) of monomers, wherein each monomer is as defined
for the compound above. Particularly, multimeric peptides such as
dendrimers may form conformational determinants or clusters due to the
presence of multiple flexible peptide monomers. In one embodiment the
compound is a dimer. In a more preferred embodiment the compound is a
dendrimer, such as four peptides linked to a lysine backbone, or coupled
to a polymer carrier, for example a protein carrier, such as BSA.
Polymerisation such as repetitive sequences or attachment to various
carriers are well-known in the art, e.g. lysine backbones, such as lysine
dendrimers carrying 4 peptides, 8 peptides, 16 peptides, or 32 peptides.
Other carriers may be lipophilic dendrimers, or micelle-like carriers
formed by lipophilic derivatives, or starburst (star-like) carbon chain
polymer conjugates.

[0135]The individual monomers may be homologous, i.e. identical to one
another, or the individual monomers may be heterologous, i.e. different
from one another. The latter type of monomers may comprise at least two
different monomers. In general dimers and multimers may comprise two or
more identical monomers, or two or more monomers different from one
another.

[0136]Pharmaceutical Composition

[0137]The invention also relates to a pharmaceutical composition
comprising one or more of the compounds defined above comprising the
third Immunoglobulin (Ig3) module, and/or the fourth Immunoglobulin (Ig4)
module, and/or the fifth Immunoglobulin (Ig5) module, and/or the first
Fibronectin III (Fn3,1) module, and/or the second Fibronectin III (Fn3,2)
module of neural cell adhesion molecule (NCAM), or a fragment, or a
variant thereof, wherein the compound is capable of interacting with an
Fibroblast Growth Factor (FGF) receptor and/or Adenosine-Tri-Phosphate
(ATP) and/or L1.

[0138]In the present context the term "pharmaceutical composition" is used
synonymously with the term "medicament".

[0139]In one embodiment the pharmaceutical composition comprises the NCAM
F3,2 module, or a fragment thereof, or a variant thereof. In another
embodiment the composition comprises the NCAM F3,1 module, or a fragment
thereof, or a variant thereof.

[0140]The compositions are preferably formulated as multimers or dimers as
discussed above.

[0142]The pharmaceutical composition may in one aspect prevent death of
cells in vitro or in vivo, wherein the composition is administered to a
subject, in vitro or in vivo in an effective amount of one or more of the
compounds described above or a composition as described below, so as to
prevent cell death of FGF receptor presenting cells and/or L1 presenting
cells in several tissues and organs as discussed herein.

[0143]The medicament of the invention comprises an effective amount of one
or more of the compounds as defined above, or a composition as defined
above in combination with pharmaceutically acceptable additives. Such
medicament may suitably be formulated for oral, percutaneous,
intramuscular, intravenous, intracranial, intrathecal,
intracerebroventricular, intranasal or pulmonal administration.

[0144]Strategies in formulation development of medicaments and
compositions based on the compounds of the present invention generally
correspond to formulation strategies for any other protein-based drug
product. Potential problems and the guidance required to overcome these
problems are dealt with in several textbooks, e.g. "Therapeutic Peptides
and Protein Formulation. Processing and Delivery Systems", Ed. A. K.
Banga, Technomic Publishing AG, Basel, 1995.

[0145]Injectables are usually prepared either as liquid solutions or
suspensions, solid forms suitable for solution in, or suspension in,
liquid prior to injection. The preparation may also be emulsified. The
active ingredient is often mixed with excipients which are
pharmaceutically acceptable and compatible with the active ingredient.
Suitable excipients are, for example, water, saline, dextrose, glycerol,
ethanol or the like, and combinations thereof. In addition, if desired,
the preparation may contain minor amounts of auxiliary substances such as
wetting or emulsifying agents, pH buffering agents, or which enhance the
effectiveness or transportation of the preparation.

[0146]Formulations of the compounds of the invention can be prepared by
techniques known to the person skilled in the art. The formulations may
contain pharmaceutically acceptable carriers and excipients including
microspheres, liposomes, microcapsules, nanoparticles or the like.

[0147]The preparation may suitably be administered by injection,
optionally at the site, where the active ingredient is to exert its
effect. Additional formulations which are suitable for other modes of
administration include suppositories, nasal, pulmonal and, in some cases,
oral formulations. For suppositories, traditional binders and carriers
include polyalkylene glycols or triglycerides. Such suppositories may be
formed from mixtures containing the active ingredient(s) in the range of
from 0.5% to 10%, preferably 1-2%. Oral formulations include such
normally employed excipients as, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, and the like. These compositions take the
form of solutions, suspensions, tablets, pills, capsules, sustained
release formulations or powders and generally contain 10-95% of the
active ingredient(s), preferably 25-70%.

[0148]Other formulations are such suitable for nasal and pulmonal
administration, e.g. inhalators and aerosols.

[0149]The active compound may be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include acid addition salts (formed
with the free amino groups of the peptide compound) and which are formed
with inorganic acids such as, for example, hydrochloric or phosphoric
acids, or such organic acids as acetic acid, oxalic acid, tartaric acid,
mandelic acid, and the like. Salts formed with the free carboxyl group
may also be derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium, or ferric hydroxides, and such organic
bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine,
procaine, and the like.

[0150]The preparations are administered in a manner compatible with the
dosage formulation, and in such amount as will be therapeutically
effective. The quantity to be administered depends on the subject to be
treated, including, e.g. the weight and age of the subject, the disease
to be treated and the stage of disease. Suitable dosage ranges are per
kilo body weight normally of the order of several hundred pg active
ingredient per administration with a preferred range of from about 0.1
μg to 5000 μg per kilo body weight. Using monomeric forms of the
compounds, the suitable dosages are often in the range of from 0.1 μg
to 5000 μg per kilo body weight, such as in the range of from about
0.1 μg to 3000 μg per kilo body weight, and especially in the range
of from about 0.1 μg to 1000 μg per kilo body weight. Using
multimeric forms of the compounds, the suitable dosages are often in the
range of from 0.1 μg to 1000 μg per kilo body weight, such as in
the range of from about 0.1 μg to 750 μg per kilo body weight, and
especially in the range of from about 0.1 μg to 500 μg per kilo
body weight such as in the range of from about 0.1 μg to 250 μg per
kilo body weight. In particular when administering nasally smaller
dosages are used than when administering by other routes. Administration
may be performed once or may be followed by subsequent administrations.
The dosage will also depend on the route of administration and will vary
with the age and weight of the subject to be treated. A preferred dosage
of multimeric forms would be in the interval 1 mg to 70 mg per 70 kg body
weight.

[0151]For most indications a localised or substantially localised
application is preferred.

[0152]Some of the compounds of the present invention are sufficiently
active, but for some of the others, the effect will be enhanced if the
preparation further comprises pharmaceutically acceptable additives
and/or carriers. Such additives and carriers will be known in the art. In
some cases, it will be advantageous to include a compound, which promote
delivery of the active substance to its target.

[0153]In many instances, it will be necessary to administrate the
formulation multiple times. Administration may be a continuous infusion,
such as intraventricular infusion or administration in more doses such as
more times a day, daily, more times a week, weekly, etc. It is preferred
that administration of the medicament is initiated before or shortly
after the individual has been subjected to the factor(s) that may lead to
cell death. Preferably the medicament is administered within 8 hours from
the factor onset, such as within 5 hours from the factor onset. Many of
the compounds exhibit a long term effect whereby administration of the
compounds may be conducted with long intervals, such as 1 week or 2
weeks.

[0154]In connection with the use in nerve guides, the administration may
be continuous or in small portions based upon controlled release of the
active compound(s). Furthermore, precursors may be used to control the
rate of release and/or site of release. Other kinds of implants and well
as oral administration may similarly be based upon controlled release
and/or the use of precursors.

[0155]As discussed above, the present invention relates to treatment of
individuals for inducing differentiation, modulating proliferation,
stimulate regeneration, neuronal plasticity and survival of FGF receptor
presenting cells or L1 presenting cells or other NCAM ligand presenting
cells in vitro or in vivo, the treatment involving administering an
effective amount of one or more compounds as defined above.

[0156]Another strategy for administration is to implant or inject cells
capable of expressing and secreting the compound in question. Thereby the
compound may be produced at the location where it is going to act.

[0157]Treatment

[0158]In a further aspect, the present invention relates to said peptides,
fragments, or variants thereof for use in the modulation of proliferation
and/or induction of differentiation and/or stimulation of regeneration,
neuronal plasticity and/or survival of cells presenting an FGF receptor.
The use is for the treatment for preventing diseases and conditions of
the central and peripheral nervous system, and of the muscles or of
various organs.

[0159]Treatment by the use of the compounds/compositions according to the
invention is in one embodiment useful for inducing differentiation,
modulating proliferation, stimulate regeneration, neuronal plasticity and
survival of cells being implanted or transplanted. This is particularly
useful when using compounds having a long term effect.

[0160]In further embodiment the treatment may be for stimulation of
survival of cells which are at risk of dying due to a variety of factors,
such as traumas and injuries, acute diseases, chronic diseases and/or
disorders, in particular degenerative diseases normally leading to cell
death, other external factors, such as medical and/or surgical treatments
and/or diagnostic methods that may cause formation of free radicals or
otherwise have cytotoxic effects, such as X-rays and chemotherapy. In
relation to chemotherapy the NCAM binding compounds according to the
invention are useful in cancer treatment of all cancer cells presenting
NCAM ligands.

[0162]Also, in relation to diseases or conditions of the muscles including
conditions with impaired function of neuro-muscular connections, such as
genetic or traumatic atrophic muscle disorders; or for the treatment of
diseases or conditions of various organs, such as degenerative conditions
of the gonads, of the pancreas, such as diabetes mellitus type I and II,
of the kidney, such as nephrosis the compounds according to the invention
may be used for inducing differentiation, modulating proliferation,
stimulate regeneration, neuronal plasticity and survival, i.e.
stimulating survival.

[0163]Furthermore, the compound and/or pharmaceutical composition may be
for preventing cell death of heart muscle cells, such as after acute
myocardial infarction, in order to induce angiogenesis. Furthermore, in
one embodiment the compound and/or pharmaceutical composition is for the
stimulation of the survival of heart muscle cells, such as survival after
acute myocardial infarction. In another aspect the compound and/or
pharmaceutical composition is for revascularisation, such as after
injuries.

[0164]It is also within the scope of the invention to use the compound
and/or pharmaceutical composition for the promotion of wound-healing. The
present compounds are capable of stimulating angiogenesis and thereby
promote the wound healing process.

[0165]The invention further discloses the use of the compound and/or
pharmaceutical composition in the treatment of cancer. NCAM regulates
motility and inhibits cancer cells from spreading.

[0166]In yet a further embodiment the use of the compound and/or
pharmaceutical composition is for the stimulation of the ability to learn
and/or of the short and/or long term memory.

[0168]A further aspect of the invention is a process of producing a
pharmaceutical composition, comprising mixing an effective amount of one
or more of the compounds of the invention, or a pharmaceutical
composition according to the invention with one or more pharmaceutically
acceptable additives or carriers, and administer an effective amount of
at least one of said compound, or said pharmaceutical composition to a
subject.

[0169]In one embodiment of the process as mentioned above, the compounds
are used in combination with a prosthetic device, wherein the device is a
prosthetic nerve guide. Thus, in a further aspect, the present invention
relates to a prosthetic nerve guide, characterised in that it comprises
one or more of the compounds or the pharmaceutical composition as defined
above. Nerve guides are known in the art.

[0170]Another aspect of the invention relates to the use of a compound as
defined above. In particular the use of a compound according to the
invention is for the production of a pharmaceutical composition. The
pharmaceutical composition is preferably for the treatment or prophylaxis
of any of the diseases and conditions mentioned above.

[0171]In yet a further aspect the invention relates to a method of
treating a disease or condition as discussed above by administering a
compound as defined herein.

[0172]Experimental

[0173]The following are non-limiting examples illustrating the present
invention.

[0174]Materials and Methods

[0175]Methods

[0176]The 15N-labelled and unlabelled protein corresponding to
aminoacids 612-705 of rat NCAM (swissprot p13596) was produced in yeast
P. pastoris. The expression product contains two N-terminal residues, A
and G, from the vector and is sequentially numbered from 1 to 96. 2 mM
unlabelled and 1 mM 15N-labelled protein (in 30 mM NaCl, 10 mM
sodium phosphate buffer, pH 7.27) were used. The 1H and 15N
resonances were assigned from spectra of DQF-COSY, TOCSY, 15N
TOCSY-HSQC NMR experiments. All data were acquired at 298 K. The NOE
constraints were derived from 80/200 ms NOESY and 125 ms
15N-NOESY-HSQC spectra with upper bounds of 2.7. 3.3 and 6.0 Å
increased by 0.5 Å if the constraint included a methyl group. 40
φ angles restraints with bounds of -120±40° and
-57±40° (derived from the 3JHNHα coupling
constants) and 4 χ1 angles (for valines) were applied. 96
structures were generated with a distance geometry/simulated annealing
protocol using the X-PLOR program. After inspection of hydrogen bond
energies, 80 hydrogen bond restraints were applied with upper bounds of 2
Å and 3 Å for the NH--O and N--O distances, respectively. All the
structures had NOE violations of less than 0.3 Å, and rms deviations
from idealized geometry for bond lengths and angles of less than 0.01
Å and 20°, respectively.

[0177]HEK293 Cell Culture and Transfection

[0178]Cells were grown in DMEM 1965 with 10% FCS, 100 U/ml penicillin, 100
μg/ml streptomycin and 58.4 g/l Glutamax. ˜0.8*106 cells
were plated in 60 mm plates and cultured for 24 hrs before being
transfected using LipofectAMIN PLUS® reagent kit according to
manufacturers instructions (Gibco BRL) with 0.2 μg PcDNA3.1(+) plasmid
encoding a his-tagged (C-terminal hexa-histidine) version of FGFR-1.
Cells were grown another 24 hrs in full medium, and then shifted to
starving media (DMEM 1965 with 0.5% FCS) overnight.

[0179]Stimulation, Purification and Western Blot Analysis

[0180]FGFR-1 transfected cells, incubated with 50 μg/ml NCAM derived
recombinant fIII.2 or FG-loop for 20 minutes or non-stimulated, were
lysed in 8M urea in PBS with 1 mM orthovanadate. The FGFR-1 was purified
from total lysate via the His-tag moiety on an IMAC column. Equal amounts
of lysate were loaded on Ni2+/NTA-sepharose (Qiagen), washed in
lysis buffer with10 mM imidazole, and his-tagged FGFR-1 was eluted in
lysis buffer with 250 mM imidazole. Samples were added SDS-PAGE sample
buffer and analysed by western blotting using anti-pentahis ab (#34660
Qiagen) or anti-phosphotyrosine ab (PY20) (#11120 Transduction
Laboratories). Bands were visualised by chemilumiscense and density was
measured using a GeneGnome from SynGene with a 16 bit camera.

[0182]To study the structural and functional properties of the second F3
module of NCAM as well as a possible interaction with the FGF-receptor,
the recombinant proteins of the second F3 module of NCAM and the second
and third Ig modules of the FGF-receptor were produced in the yeast
expression system of P. pastoris. This expression system was selected
because P. pastoris is capable of protein folding and processing similar
to higher eukaryotes, and the protein secreted into the medium can be
purified easily.

Example 1

[0183]Structure of the Second F3 Module of NCAM

[0184]The three-dimensional structure of the module was derived from 1434
experimentally determined restraints (15 restraints per residue): 1322
structurally significant nuclear Overhauser enhancement (NOE) distance
restraints (as determined by means of the program DIANA), 44 dihedral
angle restraints (40 φ and 4 χ1 ), and 68 hydrogen bond
restraints. An overlay of 30 superimposed structures for the backbone
atoms is shown in FIG. 1a. The global root mean square (rms) deviation
(rmsd) from the average of the 30 superimposed structures is 0.25 Å
for the backbone atoms and 0.68 Å for the heavy atoms. A ribbon
representation of the structure labeling the seven β-strands is
shown in FIG. 1b. The summary of the NOE statistics, energy terms and
deviations from the idealized geometry is shown in Table 1.

[0185]The structure consists of seven antiparallel β-strands arranged
in a sandwich of two β sheets, one containing three strands (ABE)
and the other four strands (GFCD). Both of the β sheets have a
right-handed twist. The triple-stranded β sheet consists of residues
Lys 7-Gly 13 (A), Ser 18-Ile 24 (B), His 59-Lys 63 (E), and the
four-stranded β sheet consists of residues Ile 33-Ala 42 (C), Ile
51, Arg 52 (D), Glu 70-Asn 79 (F) and Gly 82-Arg 92 (G). Identification
of the elements of the secondary structure was performed using the
programs PROCHECK and MOLMOL. There are two wide type β-bulges (Chan
et al., 1993) involving residues Lys 85, Ala 86 and Val 76 (G and F
β-stands), and residues Ala 77 and His 35, Tyr 36 (F and C
β-strands). The two β-bulges contribute to the right-handed
twist conformation of the four-stranded β-sheet. The β-hairpins
formed by the A and B β-strands, and the G and F β-strands are
well defined. The β-hairpin between the A and B strands corresponds
to a 4:6 type I turn with the average φ, ψ values for Glu 14 (i+1
residue) being -64±1°, 12±2°, and for Asp 15 (i+2
residue) -80±1°, 24±2°; whereas the β-hairpin
between the G and F strands corresponds to a tight 2:2 type I turn with
the average φ, ψ values for Gln 80 (i+1 residue)
-38±3°, -42±40, and for Gln 81 (i+2 residue)
-107±13°, 28±30° (Sibanda et al., 1989; Wilmont and
Thornton, 1990). The loops between the D-E and E-F strands appear to be
distorted type II turns with the average φ, ψ values for i+1
residues being -70±8°, 93±17° (Ser 55, D-E loop),
-73±20°, 154±3° (Trp 67, E-F loop), and the average
φ, ψ values for i+2 residues being 109±9°,
57±8° (Gly 56, D-E loop), 46±3°, 48±2° (Asn
68, E-F loop). The hydrophobic core, which is enclosed by the two
β-sheets, consists of residues Leu 8, Met 12, Ile 19, Val 21, Leu
23, Ile 33, Tyr 36, Val 38, Tyr 40, Ala 42, Ile 51, Leu 53, Val 60, Leu
62, Leu 65, Tyr 71, Val 73, Val 75, Ala 77, Ala 87, Phe 89, Phe 91, and
Thr 93. Interestingly, the two tryptophans Trp 47 and Trp 67, located in
the C-D and E-F loops, respectively, and Tyr 74 (F strand) are exposed on
the surface of the module, and they are not part of the hydrophobic core.

[0186]All of the 30 structures conform to the commonly applied acceptance
criteria: no violations larger than 0.5 Å for NOE restraints and
larger than 5° for dihedral angle restraints, root mean square
deviations from idealized geometry for bond lengths and bond angles less
than 0.01 Å and 2°, respectively. The quality of each
structure was assessed using the program WHAT IF. The average Z-scores
for the set of 30 structures are: -1.42±0.17 for the 2nd
generation packing quality, -1.94±0.27 for the Ramachandran plot,
-1.53±0.34 for the χ1/χ2 plot, and -2.10±0.20 for
the backbone conformation. The quality of the 30 structures was further
analyzed using the program PROCHECK. All of the analyzed main-chain and
side-chain parameters were found to be within the normal ranges when
compared to the X-ray structures of 2.0 Å resolution. The number of
residues in the most favored region of the Ramachandran plot is 75.3%.
NOE violations were analyzed using the program AQUA. The maximum NOE
violation is 0.25 Å, and the rms NOE violation is 0.0189±0.00067
Å.

Example 2

[0187]The Second F3 Module of NCAM Binds to the FGF-Receptor and ATP

[0188]Given the assignment of the NMR spectra of the module and its known
three-dimensional structure, it is possible to locate the residues that
form the binding site on the surface of the module. In the 15N-HSQC
spectrum of the 15N-labeled protein, a signal for each amino acid
with both a peptide nitrogen and proton can be observed. The changes in
the chemical shifts of the signals provide a method for the
identification of residues in a protein that are perturbed by the binding
of another molecule. To the 0.05 mM 15N-labeled sample of the second
F3 module of NCAM, 1 mM unlabeled the second or third Ig modules of the
FGF-receptor, or 5 mM AMP-PCP (an non-hydrolysable analogue of ATP) were
added. No significant changes of the chemical shifts were found in the
presence of the second Ig module of the FGF-receptor (data not shown).
The recorded changes of the chemical shifts in the presence of the third
Ig module of the FGF-receptor or ATP are shown in FIG. 2a-d. The changes
in 1H and 15N chemical shifts in the presence of the third Ig
module of the FGF-receptor were mapped onto the structure of the module
(FIG. 2e), using a cutoff of 0.006 p.p.m. and 0.03 p.p.m. for the
perturbed 1H and 15N chemical shifts, respectively.

[0189]The residues of the F3 module that experienced significant
perturbation by the third Ig module of the FGF-receptor were Tyr 36, Leu
37, Val 38, Tyr 40, Leu 53, Tyr 71, Tyr 74, Val 75, Val 76, Ala 77, Asn
79, Gln 81, Gly 82, Lys 83, Ser 84, Lys 85, Ala 87, His 88, Phe 89, Val
90 (FIGS. 2a,b). The changes of the chemical shifts of these residues
demonstrate that the presence of the third Ig module of the FGF-receptor
close to the second F3 module of NCAM alters the chemical environment at
the perturbed residues of the F3 module. The perturbed residues are
located in one well defined and coherent patch on the surface of the
module, indicating that the perturbed residues are either a part or in
the vicinity of the binding site for the interaction between the second
F3 module of NCAM and the third Ig module of the FGF-receptor (FIG. 2E).
The surface area of the perturbed residues is approximately 2600
Å2, which is significantly larger than the 1000 Å2
minimal area generally considered to be required for a biologically
specific interaction. However, the surface area of the binding site is
likely to be smaller, since some of the perturbed residues probably are
just located close to the site.

[0190]The residues of the F3 module perturbed by AMP-PCP were Tyr 74 and
Val 75 (FIG. 2c-d). The side chain of Tyr 74 is exposed on the surface of
the module and is located in the close vicinity of the ATP-binding
consensus sequence of the module: Ala 77-Glu-Asn-Gln-Gln-Gly-Lys-Ser 84
and Lys 85 (FIG. 2f). Both Lys 83 and Lys 85 are exposed on the surface
of the module and it is possible that the positively charged side chains
of Lys 83 and Lys 85 interact with the negatively charged triphosphate
moiety of ATP, whereas the side chain of Tyr 74 is involved in
hydrophobic interaction with the adenosine moiety of ATP. A possible
arrangement of the complex of ATP and the second F3 module is depicted in
FIG. 2g. The residues perturbed by ATP (Tyr 74 and Val 75) were also
perturbed by the third Ig module of the FGF-receptor, indicating that the
ATP binding site and the FGF-receptor binding site are overlapping.

Example 3

[0191]The FGF-Receptor is Activated by the Second F3 Module of NCAM and by
the FGL Peptide

[0192]Since the above NMR experiments demonstrate binding of the second F3
module of NCAM to the FGF-receptor, it was of interest to test if this
binding can induce FGF-receptor activation in living cells. Therefore,
HEK293 cells were grown for 24 h on plastic plates, and subsequently
transfected with a His-tagged version of the FGF-receptor 1 and cultured
for another 24 h. After incubation of the cells for 20 min with the below
described compounds, cells were lysed in 8M urea and the FGF-receptor was
purified from the total lysate via the His-tag moiety. The purified
FGF-receptor 1 was then analyzed by immunoblotting using antibodies
either to the His-tag or phosphotyrosine. FGF-receptor activation was
estimated by the level of the FGF-receptor phosphorylation.

[0193]From FIG. 3, it appears that addition of 5 μM second F3 module of
NCAM increased FGF-receptor phosphorylation by approx. 150% compared to
control cells. Most of the residues of the second F3 module of NCAM
perturbed by the third Ig module of the FGF-receptor are located in the
F, G β-strands and the FG turn region of the NCAM module. We
therefore tested whether a synthetic peptide spanning these residues
could variant the second F3 module in its ability to activate the
FGF-receptor. Indeed, addition of a peptide corresponding to residues Glu
72-Ala 86 (termed the FG loop peptide) at a concentration of 25 μM
also activated the FGF-receptor, increasing phosphorylation by approx.
100%, thus supporting the notion that these residues of the second F3
module are involved in the binding to the FGF-receptor (FIG. 3).

[0194]Thus, the present data demonstrate that binding of the second F3
module of NCAM to the FGF-receptor results in the activation of the
latter.

Example 4

[0195]Activation of the FGF-Receptor by the Second F3 Module of NCAM
Stimulates Neurite Growth

[0196]Because the second F3 module of NCAM and its FGF-receptor binding
part (the FG loop peptide) activate the FGF-receptor, it may be expected
that the F3 module and the FG loop peptide are capable of mimicking a
characteristic function of NCAM stimulation: neuronal differentiation as
reflected by neuritogenesis. To test this assumption, dissociated neurons
from embryonic rat hippocampus were seeded on plastic and allowed to grow
for 24 h in the presence of the below described compounds. Thereafter,
cells were fixed with paraformaldehyde, stained with Coomassie Brilliant
Blue R250 and the length of neurites was measured using a stereological
approach (Ronn et al., 2000).

[0197]As can be seen from the phase-contrast pictures (FIGS. 4a,b),
addition of the second F3 module of NCAM at a 5 μM concentration
substantially increased the length of neurites per cell as compared to
the control, non-stimulated neurons. The effect was quantified in a
dose-response study (FIG. 4c) demonstrating that the F3 module, the FG
loop peptide and a truncated version of the peptide (Ala 77-Lys 83) all
induced neurite outgrowth, with a bell-shaped dose-response curve typical
of growth factor induced neuritogenesis (Hatten et al., 1988). The
potency of the peptides was lower than that of the module, as reflected
by the fact that a 10 times higher concentration was required for maximum
effect, and the truncated form was less efficient than the extended form.
The stimulatory effect of the second F3 module and the FG loop peptide
could be abrogated by an inhibitor of NCAM-stimulated neurite outgrowth,
an antibody against the FGF-receptor. The effect of the antibody under
control conditions and on neurite outgrowth induced by the second F3
module or the FG loop, is shown in FIG. 4d. In the latter case, a
complete inhibition was achieved, further supporting the notion that the
module and the FG loop peptide interact with the FGF-receptor.

[0198]To determine the functionally important amino acids of the FG loop
peptide, the peptide was analyzed by truncations and alanine
substitutions of various amino acids. Two truncated versions (from the
N-- and C-terminal) of the FG loop peptide were produced: the nonamer Val
76-Ser 84 and the heptamer Ala 77-Lys 83. Even though the truncated
peptides were substantially shorter than the FG loop peptide, they both
retained approximately 50% of the stimulatory effect as compared to the
entire FG loop peptide (FIG. 5a), indicating that the turn region between
the F and G β-strands (Gln 80, Gln 81) and a few adjacent amino
acids from both sides of the turn are important for the interaction
between the FGF-receptor and the second F3 module of NCAM. The heptameric
peptide was subsequently analyzed by a so-called Ala-scan in which a
series of peptides, in which each amino acid sequentially was substituted
with an alanine, were tested. As can be seen from FIG. 5a, substitution
of any amino acid in the peptide resulted in a decrease of the
neuritogenic potency and a complete loss of function was achieved if Glu
78, Asn 79, Gln 80, Gly 82, Lys 83 were substituted with Ala, indicating
that these residues are important for interaction with the FGF-receptor.
Double substitution of the two amino acids from the turn region of the FG
loop (Gln 80, Gln 81) for alanines in the entire FG loop peptide also
resulted in a complete inactivation of the peptide (FIG. 5a). These
findings are corroborated by the fact that Asn 79, Gln 81, Gly 82 and Lys
83 were perturbed in the second F3 module by binding to the third module
of the FGF-receptor. However, when the residues which seem to be
important for interaction with ATP (Tyr 74, Lys 83 and Lys 85) were
substituted for alanines in the FG loop peptide, the peptide retained
about 60% of the stimulatory effect as compared to the non-mutated
peptide (FIG. 5a). The structure of the heptameric peptide in the F3
module was compared to the known three-dimensional structure of a natural
ligand of the FGF-receptor, basic FGF (PDB code: 4FGF, Eriksson et al.,
1993), and it was found that the peptide had a structure and sequence
similarity to a loop region in basic FGF, Ala 42-Arg 48. The sequence and
structure alignment of both of the peptides is shown in FIGS. 6a, b. The
heptameric peptide derived from basic FGF and a series of peptides with
Ala substitutions were tested for their capability to induce
neritogenesis, and as can be seen from FIG. 5b, the peptide derived from
basic FGF induced neurite outgrowth to the same extent as the similar
seven amino acids from the second F3 module of NCAM. Substitution of any
amino acid for alanine resulted in a complete loss of function.

[0199]Since ATP inhibited activation of the FGF-receptor by the second F3
module of NCAM (FIG. 3), it was presumed that ATP also could inhibit the
neuritogenic activity of the module. To test this assumption, neurons
were stimulated with the below described compounds in the presence of ATP
or a non-hydrolysable analogue of ATP, AMP-PCP (added at concentrations
of 0, 0.4 or 1 mM). As can be seen from FIG. 7, both ATP and AMP-PCP
substantially reduced the neuritogenic effect induced by the second F3
module and the FG loop peptide, whereas when these compounds were added
alone, they did not have any effect. In case of AMP-PCP, a complete
inhibition was achieved of the effect of both the F3 module and the FG
loop peptide, and in case of ATP, a complete inhibition was achieved only
of the effect of the FG loop, indicating that ATP is a less potent
inhibitor than its non-hydrolysable analogue AMP-PCP. Most significantly,
when the amino acid residues of the FG loop presumed to be of importance
for ATP binding (Tyr 74, Lys 83 and Lys 85) were substituted with
alanines, the peptide retained its neuritogenic potency. However, the
stimulatory effect of the peptide could no longer be inhibited by ATP
(FIG. 7), supporting the notion that ATP binding regulates interaction
between the F3 module and the FGF-receptor.

[0200]These results indicate that activation of the FGF-receptor in
neurons by the second F3 module of NCAM induces neuritogenesis and this
effect can be inhibited by ATP.

Example 5

[0201]Survival Assay for Testing Compounds of the Invention:

[0202]Cerebellar granule neurons (CGN) from 7-days old rats are grown for
7-8 days in the presence of high potassium (40 mM). Cells are washed
twice with serum-free culture medium (basal Eagle's medium BME)
containing low potassium (5 mM) and grown in serum-free medium
supplemented with FGL peptide for two days. Cultures are assayed for cell
survival (D'Mello et al., 1997; Villalba et al., 1997; Skaper et al.,
1998) by measuring reduction of MTS. MTS is a novel tetrazolium compound
(Promega, USA), which is bioreduced by cells into a formazan that is
soluble in tissue culture medium. The absorbance of the formazan at 490
nm is measured directly from 96 well assay plates without additional
processing. The conversion of MTS into the aqueous soluble formazan is
accomplished by dehydrogenase enzymes found in metabolically active
cells. The quantity of formazan product as measured by the amount of 490
nm absorbance is directly proportional to the number of living cells in
culture (Yao and Cooper, 1995);

[0203]The number of cells were determined and the amount of cells
surviving in the presence of high-concentration of potassium was set at
100%. As can be seen approx. On FIG. 8 only 60% survived in the presence
of brains-derived neurotrophic factor or basic fibroblast growth factor.
When FGL was added in a sode-range of 2-250 microgram per ml
statistically significant survival was observed up to 90% of the positive
control at a dose of 250 microgram per ml of the monomeric form of the
FGL peptide

Example 6

[0204]FG Loop Fragments and Variants

[0205]Peptides derived from the FG-lops of the neural cell adhesion
molecules L1 and NCAM (third F3-module of L1 and first F3 module of NCAM)
were prepared in different lengths, see FIG. 9a, and their effect on
neurite outgrowth from primary hippocampal neurons were tested adding the
various peptides in a concentration of 25 microM. FIG. 9b. The NCAM
peptides are referred to as FN3,1 and the L1 peptides are referred to as
L1. The variants indicated in FIG. 9a are indicated by the number of
amino acids in each peptide. As can be seen from the figure, the peptides
had a stimulatory effect on neurite outgrowth reaching statistically
significance for the nine amino acid variant of the Fgloop of first
fibronectin type III module of NCAM and the nine amino acid variant of
the FG-loop of the third fibronectin type III-module of L1.